Proteoglycans are biological molecules composed of a protein core and covalently attached side chains of glycosaminoglycans. Proteoglycans and collagen are the primary components of articular cartilage found in knee and other joints. In articular cartilage, the side chain glycosaminoglycans are principally chondroitin sulfate and keratan sulfate. The proteoglycans in articular cartilage encompass classes containing variable amounts of both chondroitin sulfate and keratan sulfate. Proteoglycans are also found in skin, tendons, cornea, sclera and elsewhere in the body and vary in the type and number of glycosaminoglycan side chains and the molecular weight of the protein core. The types of proteoglycans and their distributions have been reviewed by Poole, Biochemical Journal, 236: 1-14, (1986).
In certain human degenerative joint diseases such as osteoarthritis and rheumatoid arthritis, excessive amounts of proteoglycans are lost from cartilage, eventually leading to cartilage degeneration and loss of joint function. Proteoglycans degraded from cartilage collect in the synovial fluid of the affected joints. In osteoarthritis a variety of proteoglycans containing chondroitin sulfate and keratan sulfate are found in synovial fluid. In gout, as well as in rheumatoid arthritis, synovial fluids contain proteoglycans which conform to other proteoglycan profiles.
Physicians treating patients presenting with swollen knee joints routinely extract synovial fluid from the swollen joint and analyze the contents of the synovial fluid to diagnose the cause of swelling. Routinely, the synovial fluid is subjected to a differential white cell count and to a search for the presence of urate or calcium pyrophosphate crystals. These tests are indicators of inflammation and the presence of crystals may pinpoint one of the causes of joint fluid effusion. Routinely also, synovial fluids are subjected to a test for viscosity to assess the state of hyaluronic acid and aggregates which it may form. Sometimes such a test is also connected to formation of a hyaluronic acid "mucin clot" which occurs when acetic acid is added to synovial fluid. Hyaluronic acid is produced by synovial PG,4 lining cells and is responsible for the viscoelastic properties of synovial fluid. Its partial hydrolysis, resulting in lowered viscosity and negative "mucin clots", is one of the sequelae of inflammation. Thus routine tests of synovial fluid are directed mainly to assessing inflammatory states. This is clearly important to the patient's prognosis as prolonged joint inflammation results in cartilage destruction and attendant impairment of joint motion accompanied by pain. The most crippling of these inflammatory diseases is rheumatoid arthritis. Another form of joint destruction, probably unrelated to inflammation, is the underlying cause of osteoarthritis, a disease entity widespread among the elderly. Though there is an important need, there presently are no reliable tests which measure joint destruction. Because these degenerative diseases are chronic, patients with the diseases must be treated for an indefinite period of time. It thus becomes important to be able to monitor the effectiveness of treatment s that adjustment in treatment can be made if necessary.
Existing methods of detecting proteoglycans and glycosaminoglycans are labor-intensive, time-consuming and some are not very sensitive to small quantities of proteoglycans. Faster, easier and more sensitive methods of detecting proteoglycans and glycosaminoglycans are needed.
Saxne, et al., Annals of the Rheumatic Diseases, 45: 91-497, (1986), detected proteoglycans with a competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to human articular cartilage proteoglycan monomer were used to detect the presence of proteoglycans in synovial fluid of patient with swollen knee joints. Proteoglycan concentrations of less than 5 micrograms per milliliter were detected. Although this method is sensitive, it provides only limited information about the molecular form of the proteoglycan derived from cartilage. In addition, the assay is time-consuming, since the proteoglycans are enzymatically degraded for four hours before attaching to the solid phase and the synovial fluid test samples are enzymatically degraded for eight hours before use in the assay.
Witter et al., Arthritis and Rheumatism, 30: 519-529, (1987), detected proteoglycan degradation products in synovial fluids of patients with arthritis by separating the proteoglycans on an ion-exchange column followed by a gel permeation column and finally detected the proteoglycans in a competitive radioimmunoassay using antibodies to proteoglycans. This method is labor-intensive as well as time-consuming and unsuitable for the clinical laboratory.
Sampson et al., Analytical Biochemistry, 151: 304-308, (1985), detected glycosaminoglycans on cellulose acetate strips using .sup.125 I-labeled cytochrome c. This reagent was able to detect glycosaminoglycans at the level of 1 nanogram per 0.25 milliliters. These researchers found that .sup.125 I-cytochrome c was a more sensitive reagent for detecting glycosaminoglycans than Alcian Blue or Ponceau S which had a minimum sensitivity level of fifty nanograms per 0.25 milliliters.
Heimer et al., Analytical Biochemistry 165: 448-455 (1987) reported the detection of proteoglycans transblotted to positively charged nylon and detected with cationized .sup.125 I-cytochrome c. Using this method one nanogram of proteoglycan could be readily detected.